Light sheet generator

ABSTRACT

Systems and methods for generating light sheets suitable for use in single plane illumination microscopy may include a series of chambers in sequential communication with each other, the series of chambers including an optics system configured to convert a beam of light, such as a laser beam, into a planar light sheet. The series of chambers may include chambers having respective long axes oriented at an acute angle to each other to form a compact zig-zag pattern.

CROSS-REFERENCES

This application is based upon and claims the benefit under 35 U.S.C.§119(e) of U.S. Provisional Patent Application Ser. No. 61/927,179,filed Jan. 14, 2014.

The complete disclosure of the above-identified patent application ishereby incorporated by reference for all purposes.

FIELD

This disclosure relates to sample illumination for microscopes. Morespecifically, the disclosed embodiments relate to systems and methodsfor generation of light sheets in fields such as single planeillumination microscopy (SPIM).

INTRODUCTION

Single plane illumination microscopy (SPIM), or light sheet microscopy,utilizes a microscope objective having a first axis of observation. Alight sheet is generated to illuminate a sample, and the light sheetconsists of a collimated plane of light oriented transverse to the firstaxis. In some examples, the first axis is orthogonal to the plane of thelight sheet. The objective is typically focused at the plane of thelight sheet. In some examples, the sample is also repositioned relativeto the light sheet in order to observe various levels/planes of thesample. Multiple images may be acquired in this fashion in order tocreate a 3-D image of the sample. If the image is changing over time,multiple such 3-D image compilations may also be taken over time,resulting in so-called 4-D imaging. A light sheet generating device andmethod are described below and in the attached materials.

In the last decade, light sheet fluorescence microscopy (LSFM) hasemerged as a powerful imaging tool for cell and developmental biology.LSFM systems excite the sample with a thin light sheet and collect theresulting fluorescence along a perpendicular detection axis. Imagingvolumes are collected by sweeping the light sheet and detection planethrough the sample. As only the focal plane is illuminated at anyinstant, these microscopes provide highly efficient ‘opticalsectioning’; unlike confocal microscopes that use a pinhole to rejectbackground, little fluorescence is wasted on its way to the detector andphotodamage/bleaching are confined to the vicinity of the focal plane.Since a wide field detector (camera) is used to collect information fromthe entire imaging plane simultaneously, high signal-to-noise ratio(SNR) images may be obtained with low excitation intensities, minimizingundesirable effects like dye saturation. Collectively, these advantagesresult in instruments that are much faster, much gentler, and provideimages with much better SNR than laser scanning confocal microscopy.LSFM has been particularly beneficial in long-term 4D imaging studies,as in the embryogenesis of model organisms such as C. elegans,zebrafish, and Drosophila. Recent efforts have improved thespatiotemporal resolution of LSFM, and have enabled the study of fast,intracellular dynamics that would have been otherwise impossible tocapture with other 4D imaging systems.

Modern LSFM systems use one objective lens to deliver the excitationsheet and another to collect the fluorescence. The requiredperpendicularity between excitation and detection forces the use ofrelatively long working distance objectives and constrains the samplegeometry relative to single objective epi- or confocal fluorescencemicroscopy. Many LSFM implementations embed the sample in agarose,translating the resultant gel appropriately to the common focal point ofthe objectives. By rotating the sample, specimen views acquired atdifferent angles may be fused together into a composite volume,increasing the overall image quality by masking the effects ofscattering and light sheet degradation that plague individual views.Moreover, multiview deconvolution can be applied to compensate for thepoor axial resolution of any single view with the much better lateralresolution inherent to a corresponding perpendicular view, improvingresolution isotropy.

While appropriate for large embryos like zebrafish or Drosophila,embedding the sample in agarose is cumbersome for a large variety ofsamples that are more easily grown or deposited on conventional glasscoverslips. Inverted selective plane illumination microscopy (iSPIM) isa version of LSFM that is compatible with glass cover slips. In iSPIM,two perpendicular, water dipping objectives are placed above a samplemounted in an inverted microscope stand. A light sheet is introducedwith one objective and scanned through the sample, and the secondobjective is translated with a piezoelectric stage in order to keep theimaging plane in focus during scanning. The sample can be easily foundwith a low magnification objective mounted in the epi-fluorescence portof the inverted microscope, and translated to the focus of eachobjective. A modified version of iSPIM may be utilized to capture asecond specimen view, by alternating excitation and detection betweenthe two objectives. The resulting dual-view inverted selective planeillumination microscope (diSPIM) may provide isotropic spatialresolution (down to 330 nm) at high speed (200 images/second, 0.5seconds for a 50 plane volume).

The devices described herein may be compatible with fiber-coupled laserexcitation (making it much easier to align the excitation path, makingthe device compatible with a broad array of commercial laser excitationsources, and assuring collinearity between different excitationwavelengths). Instead of controlling multiple galvanometric mirrors,compact scan-heads (one for each specimen view) may be used to generateand sweep the excitation sheets through the sample, also aiding systemalignment. Freely available LabVIEW data acquisition software may beutilized for generating and sweeping the light sheets, controlling andsynchronizing objective piezos, and recording images from scientificcomplementary metal-oxide-semiconductor (CMOS) cameras.

SUMMARY

The present disclosure provides systems and methods for light sheetgeneration. In some embodiments, a light sheet generating system mayinclude a housing including a plurality of elongate internal chambers insequential communication with each other and containing an opticssystem. A first chamber may contain a first lens, a second chamber mayextend from the first chamber and contain a second lens, a third chambermay extend from the second chamber, and a fourth chamber may extend fromthe third chamber and contain a third lens, each chamber having arespective long axis. A light source mount may be included at a firstend of the housing, the mount configured to receive a light source anddirect a light beam down the long axis of the first chamber. The firstlens may be configured to focus the laser beam onto a first mirror. Thesecond lens may be configured to collimate the laser beam afterreflecting from the first mirror. The second mirror may be configured tosteer the collimated laser beam into a fan shape. The third lens may beconfigured to collimate the fan shaped laser into a light sheet. Thelong axes of each pair of consecutive cavities may form an acute angle.

In some embodiments, a system for conducting light sheet microscopy mayinclude a microscope having an objective including an objective axis, asample apparatus configured to hold a sample spaced from the objectivealong the objective axis, and a light sheet generating assemblyconfigured to generate a planar sheet of collimated light intersectingthe sample at an angle transverse to the objective axis. The light sheetgenerating assembly may include a laser source operatively connected toa first elongate chamber containing a first lens, a first reflectordisposed at a terminal end of the first chamber, a second elongatechamber containing a second lens and arranged at an acute angle withrespect to the first chamber, a second reflector disposed at a terminalend of the second chamber, a third elongate chamber arranged at an acuteangle with respect to the second chamber, a third reflector disposed ata terminal end of the third chamber, and a fourth elongate chambercontaining a third lens and arranged at an acute angle with respect tothe third chamber. The second reflector may include a scanning mirror.

In some embodiments, a method for generating a light sheet suitable foruse in a microscope may include generating a light beam using a lightbeam source. The beam may be directed into an optics system disposed ina housing by directing the beam into a first chamber of a series ofelongate chambers contained by the housing, a long axis of eachsuccessive chamber being oriented at an acute angle with respect to thelong axis of the immediately preceding chamber. The beam may be focused,using a first lens, onto a first mirror. The beam may be redirected,using the first mirror, toward a second lens. The beam may be collimatedusing the second lens. The beam may be redirected and fanned using ascanning second mirror. The fanned beam may be collimated using a thirdlens.

Features, functions, and advantages may be achieved independently invarious embodiments of the present disclosure, or may be combined in yetother embodiments, further details of which can be seen with referenceto the following description and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of an illustrative light sheet generationdevice in accordance with aspects of the present disclosure.

FIG. 2 is an illustrative embodiment of a light sheet generator inaccordance with aspects of the present disclosure.

FIG. 3 is another illustrative embodiment of light sheet generator inaccordance with aspects of the present disclosure.

FIG. 4 is an illustration of steps performed in an exemplary method forgenerating a light sheet for use in light sheet microscopy or the like.

DESCRIPTION Overview

Various embodiments of light sheet generation systems and methods aredescribed below and illustrated in the associated drawings. Unlessotherwise specified, a light sheet generation system and/or its variouscomponents may, but are not required to, contain at least one of thestructure, components, functionality, and/or variations described,illustrated, and/or incorporated herein. Furthermore, the structures,components, functionalities, and/or variations described, illustrated,and/or incorporated herein in connection with the present teachings may,but are not required to, be included in other light sheet generationsystems and methods. The following description of various embodiments ismerely exemplary in nature and is in no way intended to limit thedisclosure, its application, or uses. Additionally, the advantagesprovided by the embodiments, as described below, are illustrative innature and not all embodiments provide the same advantages or the samedegree of advantages.

Light sheet generation devices and systems may be interchangeably termedlight sheet scanners, light sheet generators, and/or light sheet scanunits or modules. A light sheet scanner generates a planar sheet oflight by passing a collimated laser beam through a folded optics system.The folded optics system includes a plurality of lenses, one or morefolding mirrors, and/or one or more scanning mirrors arranged togenerate a light sheet suitable for use by a SPIM (and/or iSPIM and/ordiSPIM) microscope.

A light sheet generator is shown schematically in FIG. 1, and generallyindicated at 10. Light sheet generator 10 may include a light sourceinterface 12 configured to receive a light source 14, such as a laser.Interface 12 may be configured to direct a light (e.g., laser) beam fromlight source 14 into and through a first chamber 16 within light sheetgenerator 10. For example, light source interface 12 may be coaxial withfirst chamber 16.

A series of elongate cavities or internal tunnels similar to chamber 16may be arranged within generator 10 (e.g., within a housing 17 or otherenclosure), and each successive pair of chambers may be in communicationwith each other. For example, as shown in FIG. 1, first chamber 16 maybe followed by a second chamber 18, and a third chamber 20. Optionally,a fourth chamber 22 (shown in dashed lines) may proceed from thirdchamber 20. In some examples, additional chambers may be included beyondthree or four. The respective long axis of each of the chambers may bearranged such that the chambers collectively form a zig-zag pattern.More specifically, the intersection of each pair of long axes associatedwith successive chambers may form a respective acute angle. Thisrelationship is indicated, for example, at angles 24 and 26. Such azig-zag pattern may result in a more compact device.

An optics system may be housed within light sheet generator 10. Aportion of the optics system may be disposed within or adjacent tochambers 16, 18, 20, and 22 (if present). For example, a plurality oflenses may be included. For example, first chamber 16 may include afirst lens 28, second chamber 18 may include a second lens 30, and thirdchamber 20 may include a third lens 32. Optionally, third lens 32 may belocated within fourth chamber 22, if present.

Light sheet generator 10 may further include a plurality of mirrors todirect and shape the beam as it passes through the chambers. Forexample, a first mirror 34 may be disposed at a terminal end of firstchamber 16, and a second mirror 36 may be disposed at a terminal end ofsecond chamber 36. If fourth chamber 22 is included, a third mirror 38(e.g., a fold mirror) may be included to direct the beam into the fourthchamber. Each of these mirrors may include any suitable structure ordevice configured to reflect a beam of light at a selected angle. Insome examples, the selected angle may be adjustable. In some examples,the selected angle may be altered with respect to time, such as in ascanning mirror.

An aperture 40, such as an iris or diaphragm, may be included in firstcavity 16 between light source interface 12 and first lens 28. Aperture40 may be configured to stop down the beam from light source 14, and maybe adjustable.

The optics system, including lenses 28, 30, and 32 as well as mirrors34, 36, and 38 (if present), are configured to receive a beam (e.g.,laser beam) entering the system at light source interface 12 and convertthe beam into a planar light sheet at a system exit 42. The planar lightsheet would be usable by a device 44 such as a microscope. Accordingly,light sheet generator 10 may include a microscope interface 46.Microscope interface 46 may include any suitable structure configured tofacilitate secure attachment of light sheet generator 10 to a lightreceiving portion of microscope 44. Microscope 44 may include anobjective axis, and a sample apparatus configured to hold a samplespaced from the objective along the objective axis. Light sheetgenerator 10 may be configured to generate a planar sheet of collimatedlight intersecting the sample at an angle transverse to the objectiveaxis.

Lenses 28 and 30 may be arranged as a 4f pair. In other words, adistance A from first lens 28 to first mirror 34 may be equal to thefocal length of first lens 28. Similarly, a distance B from first mirror34 to second lens 30 may be equal to the focal length of second lens 30.Accordingly, the effective distance between first lens 28 and secondlens 30 (i.e., A+B) is the sum of the respective focal lengths.Excluding other optical effects, this arrangement of the lenses resultsin a beam exiting second lens 30 that has similar or identicalcharacteristics to the beam that entered first lens 28. A similararrangement may exist between lenses 30 and 32.

First mirror 34 and/or second mirror 36 may be configured to produceselected optical effects on the beam. For example, first mirror 34 maybe configured as an anti-striping mirror (explained further below), suchthat the first mirror is caused to tilt rapidly on one or more axes byan actuator 48. In some embodiments, first mirror 34 may be stationary,functioning simply as a folding mirror to redirect the beam. In someembodiments, the angle of first mirror 34 may be adjustable.

Second mirror 36 may be configured as a scanning mirror, such that themirror tilts rapidly back and forth in two dimensions and creates afanning effect in the beam. In other words, a point created by the beamimpinging on a surface would be reflected back and forth by mirror 36such that the beam would instead create a line segment on the surface.The tilting of mirror 36 may be driven by a scanning actuator 50. Thisfanning of the beam may be collimated by third lens 32. Accordingly, thebeam remains at a selected width following the third lens, and thefanning effect does not continue beyond the third lens.

Third mirror 38, if present, may include a stationary (e.g., adjustable)reflector between scanning mirror 36 and third lens 32. Mirror 38 mayfunction to fold the beam, reflecting the beam down the length ofchamber 22.

Light sheet generator 10 may be described as compact due to the zig-zagpattern created by chambers arranged at acute angles. Generator 10 maybe described as modular because it may be separate from, and attachableto, any suitable light source and/or microscope.

Examples, Components, and Alternatives

The following examples describe selected aspects of exemplary lightsheet generators, as well as related systems and/or methods. Theseexamples are intended for illustration and should not be interpreted aslimiting the entire scope of the present disclosure. Each example mayinclude one or more distinct inventions, and/or contextual or relatedinformation, function, and/or structure.

Example 1

FIGS. 2 and 3 show illustrative light sheet scanner devices 100 and100′. Light sheet scanners 100 and 100′ are embodiments of light sheetgenerator 10, and may therefore include corresponding components. Suchcorresponding components will have corresponding names, and may beidentified below and/or indicated by appropriate reference numbers inFIGS. 2 and 3.

Light sheet scanners 100 and 100′ include similar components havingsimilar descriptions. Accordingly, both scanners will be describedsimultaneously using unprimed and primed respective reference numbers,and with selected differences being identified as needed. Light sheetscanner 100, 100′ may include a body 102, 102′ having a laser mount 104,104′ at one end, a microscope mount 106, 106′ at another end, and ahousing 108, 108′ containing an optics system 110, 110′. Body 102, 102′may include housing 108, 108′ as well as mounting hardware, adjustmentdevices, openings, threaded connections, and/or mounting surfacessuitable for containing the remaining components of scanner 100, 100′and for attaching scanner 100, 100′ to another device such as amicroscope, camera, and/or support base.

Body 102, 102′ may include a plurality of connected internal cavities orpathways (also referred to as chambers), through which a beam 103, 103′travels. Housing 108, 108′ may be constructed of rigid material, or acombination of rigid components with semi-rigid and/or resilientmaterial. Housing 108, 108′ may be configured to protect internalcomponents from the environment. For example, housing 108, 108′ may bedust-proof, water resistant, and/or resistant to mechanical shock. Insome examples, housing 108, 108′ may be configured to prevent or resistbuild up of a static electrical charge. In some examples, housing 108,108′ may be configured to exclude unwanted external light from theinternal pathways.

Laser mount 104, 104′ may include any suitable structure configured toenable attachment of a laser-generating device in a predeterminedorientation. For example, a laser mount 104 may be configured to allow acoaxially mounted laser source 112, as shown in FIG. 2. In otherexamples, such as the one shown in FIG. 3, a laser mount 104′ may beconfigured to connect with a transverse-mounted laser source 112′.

Laser mount 104, 104′ may include a collimation device 114, 114′. Inother examples, collimation device 114, 114′ may be part of or integralwith laser source 112, 112′. Collimation device 114, 114′ may includeany suitable device configured to collimate light emitted by the lasersource. In some examples, such as the one shown in FIG. 3, multiplelaser sources may be included. Collimation device 114′ may be configuredto collimate the light from all of the sources into a single collimatedbeam. Together, laser mount 104, 104′, laser source 112, 112′, andcollimation device 114, 114′ are configured to create a collimated laserbeam 103, 103′ directed down a path parallel to the long axis of a firstinternal pathway 116, 116′ in body 102, 102′.

Microscope mount 106, 106′ may include any suitable structure configuredto enable attachment of scanner 100, 100′ to a microscope or otherdevice, wherein the generated light sheet is provided to the devicethrough the mount. For example, microscope mount 106, 106′ may include astandard C-mount. For example, a typical C-mount may have a 1-inchdiameter, threaded aperture having 32 threads per inch. Other mountingdevices and arrangements may be included.

Optics system 110, 110′ may include any suitable devices and systemsconfigured to convert a collimated beam of light from a laser sourceinto a light sheet suitable for use in a SPIM microscope. In the exampleshown in FIGS. 2 and 3, optics system 110, 110′ includes an iris 118,118′, a first lens 120, 120′, a first mirror 122, 122′, a second lens124, 124′, a scan mirror 126, 126′, a fold mirror 128, 128′, and a thirdlens 130, 130′. In other examples, iris 118, 118′ may be absent and/ormore or fewer lenses and/or mirrors may be present.

Iris 118, 118′ (also referred to as an aperture, e.g., aperture 40) mayinclude any suitable structure or device configured to stop down orresize the collimated laser beam from laser source 112, 112′ by adesired amount. For example, iris 118, 118′ may include an aperture oropening in an otherwise opaque structure. In some examples, iris 118,118′ includes a diaphragm that may be adjusted to alter the size of theopening in iris 118, 118′ as desired.

First lens 120, 120′ is disposed in the path of the collimated laserbeam within first internal pathway 116, 116′. First lens 120, 120′ mayinclude any suitable lens-like optical structure configured to focus thecollimated laser beam onto first mirror 122, 122′. For example, firstlens 120, 120′ may include a compound lens. For example, first lens 120,120′ may include a doublet, such as a concave and a convex lens incontact with each other. In the example shown in FIGS. 2 and 3, firstlens 120, 120′ includes a convex and concave lens doublet with theconvex lens oriented toward the laser source. In some examples, firstlens 120, 120′ may comprise a 4f lens pair with second lens 124, 124′,as described above, such that the effective lens separation distance isapproximately equal to the sum of the focal lengths of the first andsecond lenses.

First mirror 122, 122′ may be disposed at the end (or an end portion) offirst internal pathway 116, 116′, and may be oriented to redirect beam103, 103′ down a second internal pathway 132, 132′, which may be incommunication with first internal pathway 116, 116′. First mirror 122,122′ may include any suitable device, structure, and/or surfaceconfigured to reflect the beam focused thereon by first lens 120, 120′and in so reflecting, pass the beam down the long axis of secondinternal pathway 132, 132′ toward second lens 124, 124′.

In some examples, first mirror 122, 122′ includes an adjustable, static,angled mirror. In some examples, first mirror 122, 122′ includes ascanning mirror configured to prevent or reduce striping, alternativelyreferred to as an anti-striping scan mirror and/or anti-striping mirror.Striping refers to the undesired shadowing effect of a light sheet thathas been blocked at certain points by nontransparent structures withinthe sample. This effect may be overcome or reduced by rapidlyalternating the angle of the light sheet (within the plane of the lightsheet) to essentially steer light around the obstacles, first on oneside then on the other.

Accordingly, first mirror 122, 122′ may include a scanning feature thatrapidly and symmetrically alternates the angle of the mirror. Thisscanning may be accomplished by any suitable means. In some examples,mirror scanning of mirror 122, 122′ may be effected using a suitablegalvanometer scanner to steer the moveable mirror. In some examples,mirror 122, 122′ may include a micromirror, interchangeably referred toherein as a micromirror device. In some examples including a micromirrordevice, an array of miniature or microscopic mirrors may be responsiveto an electromagnetic field, and may be controlled by applying a voltageto one or more electrodes adjacent to the array. In some preferredexamples, the micromirror device may include a single mirror rather thanan array. The micromirror device may be fabricated from a material suchas silicon using photolithographic methods similar to those used tofabricate integrated electronic circuits. In the example shown, eachmicromirror device has a single mirror that may be tilted about one ormore axes, such as two perpendicular axes, or directions by applyingappropriate electrical signals. The micromirror device may be describedas a micro-electro-mechanical system (MEMS) that may be used to reflectlight in a controlled manner.

Second lens 124, 124′ may be disposed in second internal pathway 132,132′ in the path of the beam reflected by first mirror 122, 122′. Secondlens 124, 124′ may be positioned at a distance from first mirror 122,122′ corresponding to the focal length of second lens 124, 124′. Secondlens 124, 124′ may include any suitable lens-like optical structureconfigured to collimate the beam. For example, second lens 124, 124′ mayinclude a compound lens. For example, second lens 124, 124′ may includea doublet, such as a concave and a convex lens in contact with eachother.

In the example shown in FIG. 1, second lens 124, 124′ includes a convexand concave lens doublet with the convex lens oriented away from thelaser source. As explained above, first lens 120, 120′ may comprise a 4flens pair with second lens 124, 124′. In some examples, second lens 124,124′ may comprise a 4 f lens pair with third lens 130, 130′. In someexamples, second lens 124, 124′ may comprise a first 4f lens pair withfirst lens 120, 120′ and a second 4f lens pair with third lens 130,130′.

Scan mirror 126, 126′ may be disposed at the end (or an end portion) ofsecond internal pathway 132, 132′, and may be oriented to redirect thebeam down a third internal pathway 134, 134′, which may be incommunication with second internal pathway 132, 132′. Scan mirror 126,126′ may include any suitable device, structure, and/or surfaceconfigured to reflect the beam directed thereon by second lens 124, 124′and in so reflecting, pass the beam down the long axis of third internalpathway 134, 134′ toward fold mirror 128, 128′.

Moreover, scan mirror 126, 126′ may be configured as a scanning mirror.Unlike first mirror 122, 122′, which may steer the beam to overcomestriping, scan mirror 126, 126′ may be configured to scan such that thecollimated beam is steered back and forth within a plane, in arelatively wide, fan-like manner. Accordingly, scan mirror 126, 126′ mayinclude a scanning feature that rapidly and symmetrically alternates theangle of the mirror. This scanning may be referred to as vectorscanning, and may be accomplished by any suitable means. In someexamples, mirror scanning of mirror 126, 126′ may be effected using asuitable galvanometer scanner to steer the moveable mirror. In someexamples, mirror 126, 126′ may include a micromirror device, asdescribed above regarding mirror 122, 122′.

At the other end (or end portion) of third internal pathway 134, 134′lies fold mirror 128, 128′. Fold mirror 128, 128′ may be oriented toredirect the fanned beam down a fourth internal pathway 136, 136′, whichmay be in communication with third internal pathway 134, 134′. Foldmirror 128, 128′ may include any suitable device, structure, and/orsurface configured to reflect the beam directed thereon by scan mirror126, 126′ and in so reflecting, pass the beam down the long axis offourth internal pathway 136, 136′ toward third lens 130, 130′. Foldmirror 128, 128′ may function to reduce the overall size or footprint oflight sheet scanner 100, 100′ while maintaining the effective overalllength of the optical pathway therein. Fold mirror 128, 128′ may befixed at a predetermined angle. In some examples, the angle and/orposition of fold mirror 128, 128′ may be adjustable. In some examples,mirror 128, 128′ may be omitted.

Third lens 130, 130′ may be disposed in fourth internal pathway 136,136′ in the path of the fanned beam reflected by fold mirror 128, 128′.Third lens 130, 130′ may be positioned at a distance from fold mirror128, 128′ corresponding to the focal length of third lens 130, 130′.Third lens 130, 130′ may include any suitable lens-like opticalstructure configured to collimate the beam. In other words, third lens130, 130′ may refract the fanned beam such that a collimated plane oflight, or light sheet, is created. For example, third lens 130, 130′ mayinclude a compound lens. For example, third lens 130, 130′ may include alens doublet.

Once the beam has passed through third lens 130, 130′, it is in acondition suitable for use as a light sheet in a SPIM microscope. Thelight sheet may pass through the aperture in microscope mount 106, 106′and intersect the sample as desired.

Example 2

In some embodiments, a diSPIM microscope (e.g., device 44) may utilizetwo light sheet scanners, such as light sheet generators 10, 100, and/or100′, one for each objective. Each light sheet scanner may be used in anarm portion of the microscope. The scanners may use integrated 2D MEMSmirrors to provide light weight, low vibration optical scanning of lightfrom a single mode fiber-coupled laser. The basic design of the unit mayinclude a series of 4f lens pair arrangements with the scan mirrors,apertures, and focal planes located at the foci of the lenses, asdescribed above.

The input to each light sheet scanner may include a fiber collimator(e.g., collimator 114, 114′) that accepts a single-mode FC/PC (oroptionally FC/APC) connected fiber-coupled laser source (e.g., source112, 112′). The fiber collimator may be an exchangeable part that allowssome flexibility in the focal length, and hence intrinsic beam diameter,and fiber connector type. The collimated laser beam may be stopped downwith an iris diaphragm (e.g., iris 118, 118′) if desired, therebycreating light sheets with different thicknesses that are suitable forsamples of varying size.

The collimated laser is focused onto the first fold mirror (e.g., mirror122, 122′), which can optionally be a micromirror scanner that islocated in the equivalent image optical image plane as the microscopeobjective specimen focus plane. In examples using such a scanner, thedirection of the focused laser beam may tilt about the focus point, thusproviding an anti-striping capability. After reflecting from the firstfold mirror, the focused light is collimated again and projected ontothe main 2D MEMS scanner mirror. The main scan mirror may be in theequivalent optical plane as the microscope objective back focal plane.Tilting the scan mirror thus steers the focused laser beam to differentpositions in the sample focus plane.

As suggested by the C-mount connection (e.g., mount 106, 106′), simplyconnecting scanner 10, 100, 100′ to any properly positioned microscopeC-mount camera port will allow the scanner to be used to position afocused laser spot at the microscope's sample plane. For diSPIMapplications, the camera tube lens may be positioned to image collimatedlight into the objective back focal plane so that the focused laser beamremains parallel to the optical axis when scanning off-axis.

To use the scanners to create and sweep light sheets through a sample,analog voltages may be applied to one or more/each axis of the MEMSmirrors.

Example 3

This example describes a method for generating a light sheet suitablefor use in a SPIM microscope; see FIG. 4. Aspects of the light sheetgenerators described above may be utilized in the method steps describedbelow. Where appropriate, reference may be made to previously describedcomponents and systems that may be used in carrying out each step. Thesereferences are for illustration, and are not intended to limit thepossible ways of carrying out any particular step of the method.

FIG. 4 is a flowchart illustrating steps performed in an illustrativemethod, and may not recite the complete process or all steps of themethod. FIG. 4 depicts multiple steps of a method, generally indicatedat 200, which may be performed in conjunction with a light sheetgenerator according to aspects of the present disclosure. Althoughvarious steps of method 200 are described below and depicted in FIG. 4,the steps need not necessarily all be performed, and in some cases maybe performed in a different order than the order shown.

At step 202, a beam may be generated using a light beam source, such asa laser. Step 202 may include collimating the beam after the beam isgenerated.

Step 204 includes directing the beam into an optics system disposed in ahousing containing a series of elongate chambers. Directing the beaminto the optics system may include directing the beam into a firstchamber of the series of elongate chambers. A long axis of eachsuccessive chamber may be oriented at an acute angle with respect to thelong axis of the immediately preceding chamber.

Step 206 includes focusing the beam onto a first mirror using a firstlens. The first mirror may be disposed adjacent to a distal end of afirst chamber of the series of elongate chambers.

Step 208 includes redirecting the beam with the first mirror toward asecond lens. The second lens may be disposed in a second chamber of theseries of elongate chambers. Accordingly, redirecting the beam mayinclude directing the beam down the second chamber in a directionsubstantially parallel to the long axis of the chamber. In someexamples, redirecting the beam with the first mirror includes tilting orrapidly pivoting the first mirror in an alternating fashion to reduce astriping effect in the microscope, as described above. Rapid pivoting ofthe first mirror may be performed using a micromirror device.

Step 210 includes collimating the beam with the second lens. In otherwords, the beam may exit the second lens in a condition substantiallyidentical to the condition in which it entered the first chamber. Insome examples, the second lens may form a 4f lens pair with the firstlens.

Step 212 includes redirecting and fanning the beam with a scanningsecond mirror. Scanning may include rapid tilting or pivoting of thesecond mirror to cause the beam to fan out in a selected plane. Thescanning second mirror may include a micromirror device. In someexamples, step 212 may further include reflecting the fanned laser beamtoward a third lens using a third mirror.

Step 214 includes collimating the fanned beam using the third lens tocreate a light sheet. Collimating may include refracting the fanned beamsuch that the width of the beam in the selected plane would beessentially constant following the third lens. In some examples, thethird lens may form a 4f lens pair with the second lens.

The light sheet may then be directed into or received by a microscope,such as for use in SPIM microscopy or the like.

Example 4

This section describes additional aspects and features of compact lightsheet generators, presented without limitation as a series ofparagraphs, some or all of which may be alphanumerically designated forclarity and efficiency. Each of these paragraphs can be combined withone or more other paragraphs, and/or with disclosure from elsewhere inthis application, including the materials incorporated by reference inthe Cross-References, in any suitable manner. Some of the paragraphsbelow expressly refer to and further limit other paragraphs, providingwithout limitation examples of some of the suitable combinations.

A0. A system for generating a light sheet for use in a microscope, suchas a SPIM, iSPIM, and/or diSPIM microscope, the system comprising:

a housing including a plurality of elongate internal chambers insequential communication with each other and containing an opticssystem, such that a first chamber contains a first lens, a secondchamber extends from the first chamber and contains a second lens, athird chamber extends from the second chamber, and a fourth chamberextends from the third chamber and contains a third lens, each chamberhaving a respective long axis; and

a light source mount at a first end of the housing, the mount configuredto receive a light source and direct a light beam down the long axis ofthe first chamber;

wherein the first lens is configured to focus the laser beam onto afirst mirror, the second lens is configured to collimate the laser beamafter reflecting from the first mirror, the second mirror is configuredto steer the collimated laser beam into a fan shape, and the third lensconfigured to collimate the fan shaped laser into a light sheet; and

wherein the long axes of each pair of consecutive cavities form an acuteangle.

A1. The system of paragraph A0, wherein the first mirror is ananti-striping mirror.

A2. The system of any of paragraphs A0-A1, wherein the second mirror isa scanning mirror.

A3. The system of any of paragraphs A0-A2, further including a thirdmirror between the second mirror and the third lens.

A4. The system of any of paragraphs A0-A3, further including acollimation device disposed between the laser source and the first lens.

A5. The system of any of paragraphs A0-A4, wherein the first lens andthe second lens form a 4f lens pair.

A6. The system of any of paragraphs A0-A5, wherein the first lens has afocal length, and the first lens is spaced from the first mirror by adistance equivalent to the focal length.

B0. A system for conducting light sheet microscopy comprising:

a microscope having an objective including an objective axis;

a sample apparatus configured to hold a sample spaced from the objectivealong the objective axis;

a light sheet generating assembly configured to generate a planar sheetof collimated light intersecting the sample at an angle transverse tothe objective axis;

wherein the light sheet generating assembly includes a laser sourceoperatively connected to a first elongate chamber containing a firstlens, a first reflector disposed at a terminal end of the first chamber,a second elongate chamber containing a second lens and arranged at anacute angle with respect to the first chamber, a second reflectordisposed at a terminal end of the second chamber, a third elongatechamber arranged at an acute angle with respect to the second chamber, athird reflector disposed at a terminal end of the third chamber, and afourth elongate chamber containing a third lens and arranged at an acuteangle with respect to the third chamber;

wherein the second reflector is a scanning mirror.

B1. The system of paragraph B0, wherein the first reflector is ananti-striping scanning mirror.

B2. The system of any of paragraphs B0-B1, wherein the first lens has afocal length and the first lens is positioned a distance from the firstreflector corresponding to the focal length.

B3. The system of any of paragraphs B0-B2, wherein the third reflectoris mounted at an adjustable angle with respect to the third chamber.

B4. The system of any of paragraphs B0-B3, wherein the scanning mirrorincludes a micromirror device.

B5. The system of any of paragraphs B0-B4, wherein the light sheetgenerating assembly is modular.

B6. The system of any of paragraphs B0-B5, wherein the light sheetgenerating assembly includes a housing and the elongate chambers arecontained in sequential communication within the housing.

B7. The system of paragraph B6, wherein the housing includes a lasersource interface at a first end and a microscope interface at a secondend.

B8. The system of paragraph B7, wherein the microscope interfaceincludes a C-mount.

C0. A method for generating a light sheet suitable for use in amicroscope, the method comprising:

generating a light beam using a light beam source;

directing the beam into an optics system disposed in a housing bydirecting the beam into a first chamber of a series of elongate chamberscontained by the housing, a long axis of each successive chamber beingoriented at an acute angle with respect to the long axis of theimmediately preceding chamber;

focusing the beam with a first lens onto a first mirror;

redirecting the beam, using the first mirror, toward a second lens;

collimating the beam using the second lens;

redirecting and fanning the beam using a scanning second mirror;

collimating the fanned beam using a third lens.

C1. The method of paragraph C0, further including reflecting the fannedbeam toward the third lens using a third mirror.

C2. The method of any of paragraphs C0-C1, further including reducing astriping effect in the microscope by rapidly pivoting the first mirror.

C3. The method of paragraph C2, wherein rapid pivoting of the firstmirror is performed using a micromirror device.

C4. The method of any of paragraphs C0-C3, wherein the scanning secondmirror includes a micromirror device.

C5. The method of any of paragraphs C0-C4, further including collimatingthe light beam after the beam is generated by the light beam source.

CONCLUSION

The disclosure set forth above may encompass multiple distinctinventions with independent utility. Although each of these inventionshas been disclosed in its preferred form(s), the specific embodimentsthereof as disclosed and illustrated herein are not to be considered ina limiting sense, because numerous variations are possible.

The subject matter of the invention(s) includes all novel and nonobviouscombinations and subcombinations of the various elements, features,functions, and/or properties disclosed herein. The following claimsparticularly point out certain combinations and subcombinations regardedas novel and nonobvious. Invention(s) embodied in other combinations andsubcombinations of features, functions, elements, and/or properties maybe claimed in applications claiming priority from this or a relatedapplication. Such claims, whether directed to a different invention orto the same invention, and whether broader, narrower, equal, ordifferent in scope to the original claims, also are regarded as includedwithin the subject matter of the invention(s) of the present disclosure.

I claim:
 1. A system for generating a light sheet for use in amicroscope, the system comprising: a housing including a plurality ofelongate internal chambers in sequential communication with each otherand containing an optics system, such that a first chamber contains afirst lens, a second chamber extends from the first chamber and containsa second lens, a third chamber extends from the second chamber, and afourth chamber extends from the third chamber and contains a third lens,each chamber having a respective long axis; and a light source mount ata first end of the housing, the mount configured to receive a lightsource and direct a light beam down the long axis of the first chamber;wherein the first lens is configured to focus the laser beam onto afirst mirror, the second lens is configured to collimate the laser beamafter reflecting from the first mirror, the second mirror is configuredto steer the collimated laser beam into a fan shape, and the third lensconfigured to collimate the fan shaped laser into a light sheet; andwherein the long axes of each pair of consecutive cavities form an acuteangle.
 2. The system of paragraph 1, wherein the first mirror is ananti-striping mirror.
 3. The system of paragraph 1, wherein the secondmirror is a scanning mirror.
 4. The system of paragraph 1, furtherincluding a third mirror between the second mirror and the third lens.5. The system of paragraph 1, further including a collimation devicedisposed between the laser source and the first lens.
 6. The system ofparagraph 1, wherein the first lens and the second lens form a 4f lenspair.
 7. The system of paragraph 1, wherein the first lens has a focallength, and the first lens is spaced from the first mirror by a distanceequivalent to the focal length.
 8. A system for conducting light sheetmicroscopy comprising: a microscope having an objective including anobjective axis; a sample apparatus configured to hold a sample spacedfrom the objective along the objective axis; a light sheet generatingassembly configured to generate a planar sheet of collimated lightintersecting the sample at an angle transverse to the objective axis;wherein the light sheet generating assembly includes a laser sourceoperatively connected to a first elongate chamber containing a firstlens, a first reflector disposed at a terminal end of the first chamber,a second elongate chamber containing a second lens and arranged at anacute angle with respect to the first chamber, a second reflectordisposed at a terminal end of the second chamber, a third elongatechamber arranged at an acute angle with respect to the second chamber, athird reflector disposed at a terminal end of the third chamber, and afourth elongate chamber containing a third lens and arranged at an acuteangle with respect to the third chamber; wherein the second reflector isa scanning mirror.
 9. The system of paragraph 8, wherein the firstreflector is an anti-striping scanning mirror.
 10. The system ofparagraph 8, wherein the first lens has a focal length and the firstlens is positioned a distance from the first reflector corresponding tothe focal length.
 11. The system of paragraph 8, wherein the thirdreflector is mounted at an adjustable angle with respect to the thirdchamber.
 12. The system of paragraph 8, wherein the scanning mirrorincludes a micromirror device.
 13. The system of paragraph 8, whereinthe light sheet generating assembly is modular.
 14. The system ofparagraph 8, wherein the light sheet generating assembly includes ahousing and the elongate chambers are contained in sequentialcommunication within the housing.
 15. The system of paragraph 14,wherein the housing includes a laser source interface at a first end anda microscope interface at a second end.
 16. The system of paragraph 15,wherein the microscope interface includes a C-mount.
 17. A method forgenerating a light sheet suitable for use in a microscope, the methodcomprising: generating a light beam using a light beam source; directingthe beam into an optics system disposed in a housing by directing thebeam into a first chamber of a series of elongate chambers contained bythe housing, a long axis of each successive chamber being oriented at anacute angle with respect to the long axis of the immediately precedingchamber; focusing the beam, using a first lens, onto a first mirror;redirecting the beam, using the first mirror, toward a second lens;collimating the beam using the second lens; redirecting and fanning thebeam using a scanning second mirror; collimating the fanned beam using athird lens.
 18. The method of paragraph 17, further including reflectingthe fanned beam toward the third lens using a third mirror.
 19. Themethod of paragraph 17, further including reducing a striping effect inthe microscope by rapidly pivoting the first mirror.
 20. The method ofparagraph 19, wherein rapid pivoting of the first mirror is performedusing a micromirror device.
 21. The method of paragraph 17, wherein thescanning second mirror includes a micromirror device.
 22. The method ofparagraph 17, further including collimating the light beam after thebeam is generated by the light beam source.